Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
Cell membranes must allow passage of various polar molecules, including ions, sugars, amino acids, and nucleotides. Special membrane proteins are responsible for transferring such molecules across cell membranes. These proteins, referred to as membrane transport proteins, occur in many forms and in all types of biological membranes. Each protein is specific in that it transports a particular class of molecules (such as ions, sugars, or amino acids) and often only certain molecular species of the class. All membrane transport proteins that have been studied in detail have been found to be multipass transmembrane proteins. By forming a continuous protein pathway across the membrane, these proteins enable the specific molecules to cross the membrane without coming into direct contact with the hydrophobic interior of the lipid bilayer of the plasma membrane.
There are two major classes of membrane transport proteins: carrier proteins and channel proteins. Carrier proteins bind the specific molecule to be transported and undergo a series of conformational changes in order to transfer the bound molecule across the membrane. Channel proteins, on the other hand, need not bind the molecule. Instead, they form hydrophilic pores that extend across the lipid bilayer; when these pores are open, they allow specific molecules (usually inorganic ions of appropriate size and charge) to pass through them and thereby cross the membrane. Transport through channel proteins occurs at a much faster rate than transport mediated by carrier proteins.
Channel proteins which are concerned specifically with inorganic ion transport are referred to as ion channels, and include ion channels for sodium, potassium, calcium, and chloride ions. Ion channels which open in response to a change in the voltage across the membrane are referred to as voltage-sensitive ion channels.
Voltage-sensitive sodium channels are the molecular target site of a wide variety of naturally-occurring neurotoxins (Catterall 1992). Moreover, the sodium channel is one of only a small number of insecticide target sites that has been exploited successfully to achieve effective insect control (Soderlund and Bloomquist 1989b). Three pharmacologically distinct classes of insecticides (pyrethroids/DDT analogs, N-alkylamides, and dihydropyrazoles) are known to act at three different target domains of the sodium channel (Soderlund and Knipple 1994). Moreover, at least 6 other discrete domains are implicated as sites of action for a variety of naturally-occurring neurotoxins that are known to affect sodium channel function (Soderlund and Knipple 1994); these domains may also serve as useful targets for novel insecticides. As a consequence of both the proven practical significance of the sodium channel as an insecticide target site and the rich pharmacology of its multiple neurotoxin-binding domains, the search for novel agents acting at this target continues to be an important objective of many commercial agrochemical discovery efforts.
Site-directed mutagenesis experiments with vertebrate sodium channel .alpha. subunits have identified structural domains involved in ion pore formation, channel inactivation, and the binding of some neurotoxins (reviewed in Catterall 1992). Within the pore-forming domains, two specific mutations have been identified that abolish the normal calcium-dependent blockade of sodium transport and render mutated vertebrate channels permeable to calcium (Heinemann et al. 1992) (FIG. 3).
The use of target-site based biochemical screening assays to idertify novel sodium channel ligands as leads for insecticide design would greatly enhance efforts to exploit the sodium channel as a target in insecticide discovery research. However, this strategy is presently hampered by two significant technical limitations. First, the small amounts of nervous tissue that can be obtained by dissecting individual insects make the establishment of high-throughput biochemical screens based on subcellular preparations derived from insect nervous tissue prohibitively labor-intensive. Second, the principal biochemical tools that are currently available to measure ligand-sodium channel interactions are not appropriate for screening on insect sodium channels. Specifically, the radioligand .sup.3 H!batrachotoxinin A 20-.alpha.-benzoate, which is commonly employed as a direct or allosteric probe of binding at 5 distinct sodium channel domains in mammalian brain preparations (Brown 1988), is not an effective pharmacological probe for insect sodium channels (Soderlund et al. 1989c). Also, radioisotopic ion flux assays with sodium-22 as the tracer, though useful for pharmacological characterization experiments (Bloomquist and Soderlund 1988; Ottea et al. 1989; Deecher and Soderlund 1991), are inappropriate for reasons of safety as routine high-throughput screening assays. The first limitation can be overcome by expressing insect sodium channel genes in cultured cells and employing these cells as a surrogate for native tissue in screening assays. However, overcoming the second limitation will require the development of novel methods for detecting ligand-dependent modifications of sodium channel function.